DESCRIPTION(From applicant's abstract): The long range objective of this project is to develop methods to modulate opioid peptide action through the peptidases which inactivate them. A recent effort has been made to develop a method to isolate endogenous substrates for metallopeptidases. The enkephalin degrading puromycin sensitive aminopeptidase is the initial system to be studied. Mutations which eliminate catalytic activity while having little effect on binding will be introduced into this aminopeptidase. Immobilized mutant enzyme will then be used as an affinity matrix to isolate endogenous substrates and inhibitors from tissue extracts. This technology will be extended to two other zinc containing neuropeptidases, insulysin and nardilysin. A second objective of this project will be to study a novel mechanism for regulation of the peptidase nardilysin. This enzyme contains a unique acidic domain which binds amines and either increases or decreases the reaction rate dependent on the particular substrate. Preliminary evidence suggests that nadilysin forms hetero-oligomers through binding to the acidic domain. We propose to isolate the proteins which interact with nardilysin and to study their effect on nardilysin activity as well as the activity of the interacting protein(s). The third objective focuses on the isolation and characterization of a new membrane enzyme which degrades -endorphin. This is the only membrane peptidase which acts on -endorphin, thus its tissue distribution and potential physiological role(s) will be studied. Lastly we will continue our work toward obtaining the three dimensional structure of three of the neuropeptidases, the puromycin sensitive aminopeptidase, insulysin, and nardilysin. Initial crystals of the aminopeptidase have been obtained. These structures will provide important mechanistic information and should lead to the design of potent and selective inhibitors which can be used to modulate peptidase activity.